Enzyme-catalyzed asymmetric reactions have emerged as one of the most important fields in organic and pharmaceutical synthesis. The kinetic resolution of racemic mixtures is still the most common way to get enantiomerically pure compounds on industrial scales and in academic research. Through kinetic resolution, one enantiomer of a racemic mixture is selectively reacted, whereas the other remained unreacted due to differences in transformation rates.
Immobilization of enzymes on solid supports makes them more mechanically robust, thermally stable, and easily separated from the reaction media. The catalyst activity may also be enhanced when enzymes are immobilized due to reduced enzyme aggregation, especially in non-polar organic media. Polymers and inorganic supports are typically used for immobilizing enzymes. Both covalent bonding and non-covalent interaction have been used for enzyme immobilization. The interaction between the enzyme and the support surface has a significant effect on the enzyme loading, catalyst activity, and stability against enzyme leaching during reaction.
Research has been conducted on enzyme immobilization onto mesoporous silica, mostly via physical adsorption. Porous, hydrophobic silica materials are good candidates as supports for immobilized enzymes since their hydrophobicity may facilitate the access of substrates to the entrapped enzymes within their pores. In addition, the weak hydrophobic interaction between enzyme and support allows the former to maintain its active conformation, leading to high activity. However, there still exist some challenges in the immobilization of enzymes onto hydrophobic porous silica. Conventionally, the immobilization involves stirring porous silica support with the enzyme stock. Relatively low enzyme loading is generally achieved due to the low affinity of aqueous enzyme stock solution with the hydrophobic silica support. The leaching of enzymes from the support is another problem associated with the conventional method. Blanco et al (Blanco, R. M.; Terreros, P.; Fernandez-Perez, M.; Otero, C.; Diaz-Gonzalez, G. J. Mol. Catal. B: Enzym. 2004, 30, 83) has reported that leaching of lipase from a hydrophobic mesoporous silica is not likely in anhydrous media, and the immobilized lipase remains active for 15 reaction cycles in acylation of ethanolamine with lauric acid. However, the period of each cycle is only one hour. If stirred for a longer time in anhydrous organic solvent, leaching of enzymes would be likely to occur.
There is therefore a need for a method for immobilizing an enzyme on and/or in a solid support such that leaching of the enzyme from the support is reduced. The method would preferably provide high reactivity of the enzyme and improved thermal stability of the enzyme relative to unimmobilised enzyme.